Endothelial progenitor cells (EPCs) and pluripotent stem cell-derived endothelial cells have the potential to become a reliable source of autologous cells for endothelialization of intravascular devices and vascularization of tissue engineered constructs. In order to design biomaterials that can employ EPCs to enhance endothelialization, however, a better understanding of their dynamic adhesion to material surfaces under physiological shear is needed.
Endothelial colony forming cells (ECFCs) are one type of EPCs; ECFCs are highly proliferative and are capable of forming mature and functional endothelial cells for vessel repair and postnatal angiogenesis. EPCs are a subpopulation of monocytes that are derived from myeloid cells, which are one type of leukocyte. EPCs that have been isolated from blood and expanded in vitro are frequently called late outgrowth or endothelial colony forming cells (ECFCs). Expression of surface receptors on these cells differs from other types of monocytes, “early outgrowth” EPCs, and mature endothelial cells. Advantages of ECFCs for tissue engineering applications include the relative ease and lack of comorbidity in obtaining autologous cells, the highly proliferative nature of ECFCs, and the ability of ECFCs to yield mature endothelial cells. To exploit their potential, however, it is necessary to first understand whether ECFCs behave similarly to ECs in their abilities to interact with engineered biomimetic materials and which cell surface receptors mediate these interactions.
As a precursor for endothelial cells (ECs), EPCs show an endothelial-like phenotype with high proliferative capability (especially late EPCs), and can be differentiated into mature endothelial cells that form the endothelium. Hence, potential clinical applications of EPCs include vessel repair, neovascularization of ischemic organs, and coating of vascular grafts. Previous work has shown that β2 integrins are important in EPC homing to sites of ischemia and neovascularization. Integrins also play an important role in EPC capture on EC or ECM proteins in vitro. Although the types and distribution of integrin receptors are not well identified on EPCs, prior work has estimated the number of α5β1 and αvβ3 integrin receptors present on umbilical cord-blood derived EPC using flow cytometry. Besides flow cytometry, investigation of EPC migration has shown that blocking as α5β1 decreases EPC migration. However, understanding of the constitution and function of integrins present on the membrane surface of EPCs is still very limited and needs to be further explored. Importantly, although it was known that ECFCs express as α5β1 and αvβ3 integrins, ECFCs were not known to express an integrin that would bind REDV (SEQ ID NO:2) (discussed below).
Cardiovascular disease is one of many conditions that may be treated by the insertion of stents and/or vascular grafts. However, incomplete endothelialization can reduce the effectiveness of this type of treatment. Therefore, the incorporation of endothelium specific factors, for example, tailoring biomaterials for cardiovascular implant coatings, will provide enhanced clinical treatment alternatives. One such coating includes a nanofibrous matrix that may be applied to the medical implant as a self-assembled coating. Other such materials include biomimetic materials and polymer coatings for implants and medical devices.
Previous work has attempted to identify and characterize peptides, including REDV (SEQ ID NO:2), RGDS (SEQ ID NO:3), and YIGSRG (SEQ ID NO:4), grafted on PEG hydrogels have been shown to support EPC rolling under shear. Despite the fact that REDV (SEQ ID NO:2)-grafted hydrogels reduced EPC rolling velocity the most, however, it does not support firm adhesion even at low shear rate. Thus, there is a continuing need for peptides that are capable of slowing and capturing EPCs. Notably, REDV (SEQ ID NO:2) (which binds α4β1), does not bind either α5β1 and αvβ3 integrin, which are expressed on EPCs and pluripotent stem cell-derived endothelial cells.
CRRETAWAC (SEQ ID NO:1)
The peptide CRRETAWAC (SEQ ID NO:1) has a high binding affinity and selectivity for integrin α5β1. Prior work involved constructing a heptapeptide library by ligating a synthetic oligonucleotide into fUSE 5 vector. The oligonucleotide has a core sequence of TGT(NNK)7TGT (SEQ ID NO:6) where N represents an equal molar mixture of A, C, G, T while K represents G or T. TGT was coded for cysteine and NNK was coded for all amino acid. The cysteines on each side of the peptide were designed to form disulfide bond and to form cyclic peptides. Thus, the oligonucleotide produces a library of cyclic peptides with seven random amino acids. When the peptide library was screened with α5β1 coated wells to identify the peptide with high binding affinity with α5β1, among the non-RGD containing peptides, CRRETAWAC (SEQ ID NO:1) showed the highest affinity to α5β1.
Other previous works has investigated CRRETAWAC (SEQ ID NO:1) in endothelialization of expanded polytetrafluoroethylene (ePTFE) surfaces by incorporating GSSSCRRETAWAC (SEQ ID NO:7) (FIG. 4). When ePTFE was modified with GSSSCRRETAWAC (SEQ ID NO:7), it supported EC attachment and proliferation under static conditions. Furthermore, a significant lower coverage of the surface by platelets was observed comparing to RGD surface and FN-coated glass. More notably, use of this peptide for capture of cells from physiological flow conditions has not previously been considered.
—(C16)2-Glu-C2-KSSPHSRNSGSGSGSGSGRGDSP (SEQ ID NO:8) (PR_b) for α5β1
Despite the fact that PRb contained the ubiquitous RGD peptide and the synergistic PHSRN (SEQ ID NO:9) peptide, this peptide is chosen to test for the capability on EPC rolling due to the its superior design in accurately mimicking FN's binding affinity for α5β1. This peptide has been shown to mimic FN through HUVEC adhesion, spreading, and extracellular FN production (Mardilovich et al., 2006). In native FN, the distance between PHSRN (SEQ ID NO:9) and RGD is 30-40 Å which is a critical factor for PHSRN (SEQ ID NO:9) to perform its synergistic role in adhesion. With the length for each amino acid being 3.7 Å, the Kokkoli Lab used SGSGSGSGSG (SEQ ID NO:10), which is a total of 10 amino acids to give a linker distance of 37 Å, to match the distance between PHSRN (SEQ ID NO:9) and RGD. In addition, it has been shown that the ratio of hydrophilic to hydrophobic residues in between PHSRN (SEQ ID NO:9) and RGD in FN is almost 1:1 (Mardilovich & Kokkoli, 2004). The repeating SG sequence was chosen to mimic this ratio. Therefore, PRb was able to mimic the adhesion property of FN for α5β1.
HSDVHK (SEQ ID NO:11) (P11)
P11 was discovered by screening through PS-SPCL using protein-protein competitive inhibition assay. The PS-SPCL that was studied was comprised of 114 types of hexapeptide mixture and this library was divided into 6 groups with each group has various amino acids residues at each position. The PS-SPCL together with fluorescently labeled vitronectin (VN) were added onto αvβ3-coated surface and allowed to compete for the αvβ3 integrin. As shown in FIG. 5, peptides with histidine, H, at Position 1 showed the lowest fluorescent intensity meaning it was highly competitive for αvβ3 and inhibited the fluorescently labeled VN to bind αvβ3. Similarly, histidine at Position 5 and lysine, K, at position 6 showed the same result. On the other hand, more than one amino acid showed similar result at Position 2, 3, and 4. Therefore, 12 hexapeptides with sequence HXXXHK (SEQ ID NO:13) were synthesized where X represents glycine/histidine/serine at Position 2, aspartic acid/leucine at Position 3, and leucine/valine at Position 4. These 12 hexapeptides were further screened using the same competitive inhibition assay and HSDVHK (SEQ ID NO:11) was found to be the most competitive for αvβ3. Thus, HSDVHK (SEQ ID NO:11) has a high specificity and affinity for αvβ3 and it has high potential in capturing αvβ3-expressing EPCs.
NCKHQCTCIDGAVGCIPLCP (SEQ ID NO:12) (V2)
V2 is a peptide representing the 116-135 residues of the cysteine-rich heparinbinding protein (CCN1). The functions of CCN1 include regulating cell adhesion, migration, proliferation, survival and differentiation in mesenchymal cells. Studies have shown that CCN1 binds directly to αvβ3 and mediates pro-angiogenic activities. Previous studies have evaluated peptides of different portion of CCN1 and reported that V2 was responsible portion for HUVEC adhesion specifically through αvβ3. Previous work has also shown that when the D residue was altered into A, the altered V2 peptide lost the capability to bind HUVEC through αvβ3 integrin. Furthermore, shorter versions of V2 have also shown similar results meaning V2 is the exact peptide required to bind αvβ3. Therefore, V2 is a potential peptide to capture EPCs due to its specific binding to αvβ3.